Oral administration of pneumococcal antigens

ABSTRACT

Oral or peroral administration, including intragastrically, of killed whole pneumococci, lysate of pneumococci and isolated and purified PspA, as well as immunogenic fragments thereof, particularly when administered with an adjuvant such as cholera toxin provides protection in a host, animal or human, against pneumococcal infection, including colonization, and systemic infection, such as sepsis. The ability to elicit protection against pneumococcal colonization in a host prevents carriage among immunized individuals, which can lead to elimination of disease from the population as a whole.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/482,981, filed Jun. 7, 1995, which in turn is a continuation-in-partof application Ser. No. 08/458,399, filed Jun. 2, 1995, incorporatedherein by reference.

Application Ser. No. 08/482,981 is also a continuation-in-part ofapplication Ser. No. 08/446,201, filed May 19, 1995 as acontinuation-in-part of copending U.S. patent application Ser. No.08/312,949, filed Sep. 30, 1994, which in turn is a continuation-in-partof application Ser. No. 08/246,636, filed May 20, 1994.

Reference is also made to U.S. patent application Ser. No. 08/048,896filed Apr. 20, 1993, as U.S. application Ser. No. 08/246,636 is itselfis a continuation-in-part of copending U.S. patent application Ser. No.08/048,896 filed Apr. 20, 1993, which itself is a continuation-in-partof copending U.S. patent application Ser. No. 07/835,698 filed Feb. 12,1992, which itself is a continuation-in-part of U.S. patent applicationSer. No. 07/656,773 filed Feb. 15, 1991 (now abandoned). The disclosureof each of such applications is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to oral immunization or administration of hosts,animals or humans, with pneumococcal antigens to stimulate animmunological response and preferably provide protection againstpneumococcal infection, preferably against colonization, and morepreferably against systematic and humoral infection; and, tocompositions therefor.

Several publications are referenced in this application. Full citationto these references is found at the end of the specification immediatelypreceding the claims or where the publication is mentioned; and each ofthese publications is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae causes more fatal infections world-wide thanalmost any other pathogen (Anonymous, 1991; Fraser, 1982). In theU.S.A., deaths caused by S. pneumoniae rival in numbers those caused byAIDS (Anonymous, 1991). In the U.S.A., most fatal pneumococcalinfections occur in individuals over 65 years of age, in whom S.pneumoniae is the most community-acquired pneumonia. In the developedworld, most pneumococcal deaths occur in the elderly, or inimmunodeficient patents including those with sickle cell disease. In theless-developed areas of the world, pneumococcal infection is one of thelargest causes of death among children less than 5 years of age (Bermanet al., 1985; Greenwood et al., 1987; Spika et al., 1989, Bale, 1990).The increase in the frequency of multiple antibiotic resistance amongpneumococci and the prohibitive cost of drug treatment in poor countriesmake the present prospects for control of pneumococcal diseaseproblematical (Munoz et al., 1992; Marton et al., Klugman, 1990).

Humans acquire pneumococci through aerosols or by direct contact.Pneumococci first colonize the upper airways and can remain for weeks ormonths. As many as 50 or more of young children and the elderly arecolonized. In most cases, this colonization results in no apparentinfection (Gray et al., 1980; Gray et al., 1981; Hendley et al., 1975).Studies of outbreak strains have suggested that even highly virulentstrains can colonize without causing disease (Smillie et al., 1938;Smillie et al., 1936; Gratten et al., 1980; DeMaria et al., 1984). Theseexpectations have been recently confirmed using molecular probes tofingerprint individual clones (M. J. Crain, personal communication toone of the inventors). In some individuals, however, the carriedorganism can give rise to symptomatic sinusitis or middle earinfections. If pneumococci are aspirated into the lung, especially withfood particles or mucus, they can cause pneumonia. Infections at thesesites generally shed some pneumococci into the blood where they can leadto sepsis, especially if they continue to be shed in large numbers fromthe original focus of infection. Pneumococci in the blood can reach thebrain where they can cause meningitis. Although pneumococcal meningitisis less common than other infections caused by these bacteria, it isparticularly devastating; some 10% of patients die and greater than 50%of the remainder have life-long neurological sequelae (Bohr et al.,1985; Klein et al., 1981).

In elderly adults, the present 23-valent capsular polysaccharide vaccineis about 60% effective against invasive pneumococcal disease withstrains of the capsular types included in the vaccine (Bolan et al.,1986; Shapiro et al., 1991). The 23-valent vaccine is not effective inchildren less than 2 years of age because of their inability to elicitadequate responses to most polysaccharides (Cowan et al., 1978;Gotschlich et al., 1977). Improved vaccines that can protect childrenand adults against invasive infections with pneumococci would helpreduce some of the most deleterious aspects of this disease. A vaccinethat protected against disease but did not reduce pneumococcal carriagerates would not, however, be expected to control the disease inimmunocompromised individuals (Shapiro et al., 1991) and in unimmunizedindividuals. Such a vaccine would also not be expected to affect therates of infection in immunized children prior to the development of anadequate antibody or immunological response.

A strategy that could control infections in all of these individualswould be any form of immunization that prevented or greatly reducedcarriage, and hence transmission of pneumococci. In the case ofimmunization of young children with Haemophilus influenzae group bpolysaccharide-protein conjugates, it has been observed that carriage isreduced from about 4% to less than 1%, (Barbour et al., 1993), apossible explanation of concomitant herd immunity (Chiu et al., 1994).If a vaccine could prevent colonization by pneumococci, such a vaccinewould be expected to prevent virtually all pneumococcal infections inthe immunized patients. Since even unimmunized patients must acquirepneumococci from others, a vaccine that reduced carriage should reduceinfections in immunocompromised, as well as unimmunized patients. Infact, an aggressive immunization program, coupled with antibiotictreatment of demonstrated carriers, might be able to largely eliminatethe human reservoir of this organism. It may not be possible, however,to totally eliminate pneumococci since there are a number of reportsthat they have been found in laboratory rodents (Fallon et al., 1988).Whether these pneumococci are infectious for man, easily transmittableto man, or even pathogens in wild rodents is not known. S. pneumoniaedoes not live free in the environment.

Although intramuscular immunization with capsular polysaccharidevaccines has been effective at reducing the incidence of pneumococcalsepsis in the elderly (Shapiro et al., 1991), it has not been reportedto affect pneumococcal carriage rates in children up to 54 months of age(Douglas et al., 1986; Douglas et al., 1984). The principal determinantof specific immunity at mucosal surfaces is secretory IgA (S-IgA) whichis physiologically and functionally separate from the components of thecirculatory immune system. Mucosal S-IgA response are predominantlygenerated by the common mucosal immune system (CMIS) (Mestecky, 1987),in which immunogens are taken up by specialized lympho-epithelialstructures collectively referred to as musoca-associated lymphoid tissue(MALT). The term common mucosal immune system refers to the fact thatimmunization at any mucosal site can elicit an immune response at allother mucosal sites (Mestecky, 1987). Thus, immunization in the gut canelicit mucosal immunity in the upper airways and visa versa. The beststudied MALT structures are the intestinal Peyer's patches (Mestecky,1987). Further, it is important to note that oral immunization caninduce an antigen-specific IgG response in the systemic compartment inaddition to mucosal IgA antibodies (McGhee, J. R. and H. Kiyono 1993,"New perspectives in vaccine development: mucosal immunity toinfections", Infectious Agents and Disease 2:55-73).

Most soluble and non-replicating antigens are poor mucosal immunogens,especially by the peroral route, probably because they are degraded bydigestive enzymes and have little or no tropism for the gut associatedlymphoid tissue (GALT). A notable exception is cholera toxin (CT). CT isa potent mucosal immunogen (Elson, 1989; Lycke et al., 1986; Wilson etal., 1989) probably because of the GM1 ganglioside-binding property ofits binding subunit, CTB, that enables it to be taken up by the M cellsof Peyer's patches and passed to the underlying immunocompetent cells.In addition to being a good mucosal immunogen, CT is a powerful adjuvantwhich greatly enhances the mucosal immunogenicity of other solubleantigens co-administered with it (Elson, 1989; Lycke et al., 1986;Wilson et al., 1989). Although it remains somewhat controversial, pureor recombinant CTB probably does not have these properties whenadministered intragastrically (i.g.) as an adjuvant. Very small amounts(<1 μg) of intact CT, however, can act synergistically with CTB as apowerful oral adjuvant (Wilson et al., 1990). This finding may accountfor apparent adjuvant activity of many commercial preparations of CTBthat usually contain small amounts of contaminating CT.

The mechanisms by which CT and CTB act as adjuvants are not fullyunderstood, but are certainly complex, and appear to depend on severalfactors including 1) the toxic activity associated with theADT-ribosylating property of the Al subunit (Abbas et al., 1991); 2)increased permeability of mucosae (Abbas et al., 1991; Ziegler-Heitbrocket al., 1992); 3) enhanced antigen-presenting cell function (withincreased levels of IL-1) (Abbas et al., 1991; Ziegler-Heitbrock et al.,1992); as well as 4) direct stimulation of T and B cell activities(Elson, 1989; Lycke et al., 1986; Wilson et al., 1989; Wilson et al.,1990). This last point is controversial, however, as the in vitroeffects of CT or CTB on T and B cells are generally inhibitory ratherthan stimulatory (Abbas et al., 1991). Nevertheless, numerous reportsattest to the in vivo mucosal immunoenchancing effects of CT and of CTBcoupled to antigens (Hakansson et al., 1994; Anderson et al., 1981;Liang et al., 1988; Dagen et al., 1995; Dillard et al., 1994; Szu et al.1989). Recent studies have shown that orally-administered CT can induceTh2 type responses for antigen-specific serum IgG and mucosal IgAantibodies (Xu-Amano, J., H. Kiyono, R. J. Jackson, H. F. Staats,Fujihashi, P. D. Burrows, C. O. Elson, S. Pillai and J. R. McGhee. 1993,"Helper T cell subsets for immunoglobulin A responses: Oral immunizationwith tetanus toxoid and cholera toxin as adjuvant selectively inducesTh2 cells in mucosa associated tissues", J. Exp. Med. 178:1309-1320,Xu-Amano, R. J. Jackson, K. Fujihashi, H. Kiyono, H. F. Staats and J. R.McGhee, 1994, "Helper Th1 and Th2 cell responses following mucosal orsystemic immunization with cholera toxin", Vaccine 12:903-911). RecentlyElson et al. have shown that CT selectively inhibits CD8⁺ cells, andtherefore tends to abrogate suppressive-effects (Elson et al., 1995).

Since immunity to carriage would be expected to operate at the mucosalsurface, any attempt to identify antigens for vaccines against carriageshould include immunizations designed to elicit mucosal immuneresponses. Accordingly, the oral (including peroral, intragastric)immunization or administration with pneumococcal proteins, as in thepresent invention has not, it is believed, been heretofore disclosed orsuggested or, in addition, the evaluation of adjuvants, as in thepresent disclosure, has not, it is believed, been heretofore taught orsuggested.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now been surprisinglyfound that oral or peroral administration, preferably into the gut(e.g., stomach, intestines, digestive tract; intragastric), ofpneumococcal surface protein A (PspA) or an immunogenic fragment thereofelicits an immunological response and can even provide protection to ahost against pneumococcal infection such as colonization and/or systemicinfection.

Accordingly, in one aspect, the present invention provides a method ofprotecting a host, preferably a human host, against pneumococci and/orsystemic infection by oral or peroral administration to the host,preferably by administration into the gut (stomach, intestines,digestive tract; e.g., intragastrically) of the host, of an effectiveamount of at least one pneumococcal surface protein A (PspA) and/or animmunogenic fragment thereof containing at least oneprotection-eliciting epitope.

In another aspect, the present invention provides a method of elicitingan immunological response in a host against pneumococci and/or systemicinfection by oral or peroral administration to the host, preferablyadministration into the gut (stomach, intestines, digestic tract; e.g.,intragastrically) of the host, of an effective amount of at least onePspA and/or an immunogenic fragment thereof containing at least oneepitope. More preferably, the response is protective and the epitope isprotection-eliciting.

The PspA may be in the form of killed whole S. pneumoniae or a lysate ofwhole S. pneumoniae. Alternatively, the PspA may be in the form ofpurified isolated protein or a fragment thereof (individually and/orcollectively, for purposes only of shorthand in this specification,"PspA") may be obtained from bacterial isolates or may be formedrecombinantly. The PspA can be from in vivo expression thereof by asuitable vector containing DNA coding for PspA by recombinanttechniques. The PspA is preferably in a vaccine or immunogeniccomposition. Such a composition can include a pharmaceuticallyacceptable adjuvant and/or a pharmaceutically acceptable carrier.

The PspA may be mixed with pharmaceutically acceptable excipients whichare compatible with the PspA. Such excipients may include water, saline,dextrose, glycerol, ethanol, and combinations thereof. The immunogeniccompositions and vaccines may further contain auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness thereof.

In a preferred aspect of the invention, the PspA is administered withnon-toxic amounts of cholera toxin as an adjuvant.

The oral administration preferably is effected by delivery into thestomach or gut, i.e., intragastrically to provide protection to the hostagainst infection, preferably colonization, and more preferably againstsystemic infection. The oral administration also can provide protectionto the host against pulmonary infection as well as protection to thehost against an infection starting as a pulmonary infection. However,the oral administration can also involve respiratory mucosa, gingivalmucosa or alveolar mucosa, and the administration can be perlingual orsublingual or into the mouth or respiratory tract, especially if thecomposition is administered in a liquid form, e.g., as a syrup, elixiretc. However, intragastric administration is preferred.

Thus, compositions of the invention especially for oral administration,are conveniently provided as liquid preparations, e.g., isotonic aqueoussolutions, suspensions, emulsions, or viscous compositions which may bebuffered to a select pH. However, since delivery to the digestive tractis preferred, compositions of the invention can be "solid" from thepills, tablets, capsules, caplets, and the like, including "solid"preparations which are time-release or which have a liquid filling,e.g., gelatin covered liquid, whereby the gelatin is dissolved in thestomach and/or small intestine for delivery into the gut and/ordigestive system.

In a particular aspect of the invention, there is provided a method ofimmunization of a host against Streptococcus pneumoniae which comprisesorally or perorally administering to the host an immunizing amount ofpneumococcal surface protein A (PspA) in the form of a killed wholepneumococci, a lysate of pneumococci or an isolated PspA or animmunogenic fragment thereof.

The present invention further provides a vaccine composition orimmunogenic composition for oral or peroral administration to a host,preferably for administration into the gut (stomach, digestive tract;e.g., intragastrically) of a host to confer protection or elicit animmunological response, against S. pneumoniae, which comprises:

an effective amount of a pneumococcal surface protein A (PspA) in theform of a killed whole pneumococci, a pneumococcal lysate, an isolatedand purified PspA or an immunogenic fragment thereof containing at leastone epitope, preferably protection-eliciting epitope, and optionally

an adjuvanting amount of an adjuvant, preferably CT, and optionally

a pharmaceutical carrier therefor.

Moreover, the present invention provides a compartmentalized kitcomprising PspA or a fragment thereof, and a suitable carrier, diluentor excipient; and optionally, a pharmaceutically acceptable adjuvant;and further optionally, instructions for admixture and/oradministration.

Other objects and embodiments of the invention are disclosed in or areobvious variants from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following description, reference is made to the accompanyingFigures, wherein:

FIGS. 1 and 2 show bar graphs (Reciprocal Log2 ELISA (enzyme linkedimmunoadsorbant assay) Titers v. Days after immunization) of IgM, IgGand IgA responses of mice orally-immunized by gastric intubation with7.5 μg of purified native PspA, with (FIG. 2) and without (FIG. 1) 10μg/mouse CT, 7, 14, 21, 28, 35, 42 and 49 days after immunization (openbars=IgA, darkened bars=IgM, bares with diagonal stripes=IgG) (Log2=4 isjust below the limited detection of the assay. This, values of 4 shouldbe considered as indicating that there was no detective antibody);

FIG. 3 shows a time course of PspA-specific serum antibody responses inmice subcutaneously immunized with 0.5 μg of PspA and CFA (antibodytiters were examined in pooled sera collected from 6 mice by ELISA);

FIG. 4 shows the elucidation of PspA-specific antibody forming cells(AFCs) in spleen of mice simultaneously immunized with 0.5 μg of PspAand CFA (AFCs were assessed on 14 days after 2nd immunization by theELISPOT assay);

FIG. 5 shows the kinetics of antigen-specific serum antibody responsesin mice orally immunized with PspA ((a) mice were orally immunized with7.5 μg of PspA and (b) 7.5 μg of PspA together with 10 μg of CT.Antibody titers were examined in pooled sera collected from 6 mice bythe ELISA);

FIG. 6 shows an analysis of antigen-specific fecal IgA responses in miceorally immunized with 7.5 μg of PspA together with 10 μg of CT (antibodytiters were examined in pooled fecal extracts collected from 6 mice bythe ELISA); and

FIG. 7 shows the time cource of PspA-specific IgG subclass in miceorally immunized with 7.5 μg of PspA plus 10 μg of CT (antibody titerswere examined in pooled sera collected from 6 mice by the ELISA).

DETAILED DESCRIPTION OF INVENTION

As discussed above, the principal determinant of specific immunity atmucosal surfaces is secretory IgA (S-IgA) which is physiologically andfunctionally separate from the components of the circulatory immunesystem. S-IgA antibody responses may be induced locally by theapplication of suitable immunogens to a particular mucosal site. Thebulk of mucosal S-IgA responses, however, are the results of immunitygenerated via the common mucosal immune system (CMIS) (Mestecky, 1987),in which immunogens are taken up by specialized lympho-epithelialstructures, collectively referred to as mucosa-associated lymphoidtissues (MALT). The best studied immunologic lymphoepithelial structuresare the gut-associated lymphoid tissues (GALT), such as intestinalPeyer's patches (Mestecky, 1987). Other structurally and functionallysimilar lymphoid follicles occur at other mucosal surfaces, includingthose of the respiratory tract (Croitoru et al., 1994).

Bronchus-associated lymphoid tissue (BALT) was described by Bienenstock(Bienenstock et al., 1973a; Bienenstock et al. 1973b) in experimentalanimals, but is apparently not present in the noninfected humanbronchial tree (Pabst, 1992). The upper respiratory tract in humans,however, is furnished with Waldeyer's ring of tonsils and adenoids. Inrodents, the functional equivalent of these consists of nasal-associatedlymphoid tissue (NALT), a bilateral strip of lymphoid tissue withoverlying M-like epithelial cells at the base of the nasal passages(Kuper et al., 1992).

According to the present invention, a host can be effectively immunizedby oral instillation (e.g., intragastric instillation) of bacterialprotein immunogens, preferably PspA or a fragment thereof, preferablymixed with an adjuvant, more preferably cholera toxin (CT). Of course,as an adjuvant, the amount of cholera toxin used is non-toxic to thehost.

The ability of a vaccine to protect against pneumococcal colonization,as provided herein, means that the active component may protect againstdisease not only in the immunized host but, by eliminating carriageamong immunized individuals, the pathogen and hence any disease itcauses may be eliminated from the population as a whole.

Oral or peroral administration (e.g., intragastric administration) canalso prevent sepsis resulting from administration of pneumococci, sothat the vaccine can protect against both pneumococcal colonization andsepsis (systemic infection).

As mentioned, PspA is the preferred antigen. WO 92/14488 is incorporatedherein by reference. In published International patent application WO92/14488, there are described the DNA sequences for the PspA gene fromS. pneumoniae Rx1, the production of a truncated form of PspA by geneticengineering and the demonstration that such truncated form of PspAconfers protection in mice to challenge with live pneumococci.

From sequences of the PspA gene, it has been shown that PspA proteinsare variable in size (roughly 70 kDa). The C-terminal 37% of themolecule is largely composed of the 20-amino acid repeats which form abinding site that permits PspA to attach to the phosphchloine residuesof the pneumococcal lipoteichoic acids. The central region of PspA isrich in prolines and is suspected to be the portion of the molecule thatpasses through the cell wall. The sequence of the N-terminal 80% of themolecule is largely β-helical and contains the region of PspA that canelicit antibodies that are protective against sepsis. Although PspA'sare almost always at least slightly different from one another, there isenough cross-reactivity between them that antibodies or an immunologicalresponse to one PspA detect or is effective with respect to PspAs on allpneumococci. Moreover, immunization with one PspA can either protectagainst death or delay death with virtually all different challengestrains. Accordingly, a mixture of a small number of PspA's couldeffective immunity against most pneumococci.

The immunoprotective truncated PspAs described in WO 92/14488 may beused in the present invention as the PspA fragments described above fororal or peroral administration.

Numerous vector systems for in vitro and in vivo expression ofrecombinant proteins are known; e.g., bacterial systems such as E. coli;and virus systems such as bacterial viruses, poxvirus (vaccinia, avipoxvirus, e.g., canarypox virus, fowlpox virus), baculovirus, herpes virus;yeast; and the like; and, these systems may be used for producingrecombinant PspA using the coding therefor or of WO 92/14488.

Immunogenicity can be significantly improved if the antigen (PspA) isco-administered with an adjuvant, commonly used as 0.001% to 50% percentsolution in phosphate buffered saline. Adjuvants enhance theimmunogenicity of an antigen (PspA) but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system. Adjuvants can also attractcells of the immune system to an antigen depot and stimulate such cellsto elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune response to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccarides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are typically non-covalently linked toantigens and are formulated to enhance the host immune response.Aluminum hydroxide and aluminum phosphate (collectively commonlyreferred to as alum) are routinely used as adjuvants in human andveterinary vaccines. The efficacy of alum in increasing antibodyresponses to diphtheria and tetanus toxoids is well established and,more recently, a HBsAg vaccine has been adjuvanted with alum.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's complete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes. Toefficiently induce humoral immune response (HIR) and cell-mediatedimmunity (CMI), immunogens are preferably emulsified in adjuvants.

Desirable characteristics of ideal adjuvants include any or all of:

(1) lack of toxicity;

(2) ability to stimulate a long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) ability to elicit both CMI and HIR to antigens administered byvarious routes;

(5) synergy with other adjuvants;

(6) capability of selectively interacting with populations of antigenpresenting cells (APC);

(7) ability to specifically elicit appropriate T_(H) 1 or T_(H) 2cell-specific immune responses; and

(8) ability to selectively increase appropriate antibody isotype levels(for example IgA) against antigens.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al. on Aug. 8, 1989 whichis incorporated herein by reference thereto teaches glycolipid analogsincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates,each of which is substituted in the sugar residue by an amino acid, asimmune-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No.4,855,283) reported that N-glycolipids analogs displaying structuralsimilarities to the naturally occurring glycolipids, such asglycosphingolipids and glycoglycerolipids, are capable of elicitingstrong immune responses in both herpes simplex virus vaccine andpseudorabies virus vaccine. Some glycolipids have been synthesized fromlong chain alkylamines and fatty acids that are linked directly with thesugar through the anomeric carbon atom, to mimic the functions of thenaturally occurring lipid residues.

U.S. Pat. No. 4,258,029 granted to Moloney, assigned to ConnaughtLaboratories Limited and incorporated herein by reference thereto,teaches that octadecyl tyrosine hydrochloride (OTH) functions as anadjuvant when complexed with tetanus toxoid and formalin inactivatedtype I, II and III poliomyelitis virus vaccine. Octodecyl esters ofaromatic amino acids complexed with a recombinant hepatitis B surfaceantigen, enhanced the host immune responses against hepatitis B virus.

As previously mentioned, compositions of the invention, especially fororal administration, are conveniently provided as liquid preparations,e.g., isotonic aqueous solutions, suspensions, emulsions or viscouscompositions which may be buffered to a selected pH. However, sincedelivery to the digestive tract is preferred, compositions of theinvention can be in the "solid" form of pills, tablets, capsules,caplets and the like, including "solid" preparations which aretime-released or which have a liquid filling, e.g., gelatin coveredliquid, whereby the gelatin is dissolved in the stomach and/or smallintestine for delivery to the gut and/or digestive system.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing. The viscouscompositions may be in the form of gels, lotions, ointments, creams andthe like and will typically contain a sufficient amount of a thickeningagent so that the viscosity is from about 2500 to 6500 cps, althoughmore viscous compositions, even up to 10,000 cps may be employed.Viscous compositions have a viscosity preferably of 2500 to 5000 cps,since above that range they become more difficult to administer.However, above that range, the compositions can approach solid orgelatin forms which are then easily administered as a swallowed pill fororal ingestion.

Liquid preparations are normally easier to prepare than gels and otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially toanimals, children, particularly small children, and others who may havedifficulty swallowing a pill, tablet, capsule or the like, or inmulti-dose situations. Viscous compositions, on the other hand can beformulated within the appropriate viscosity range to provide longercontact periods with mucosa, such as the lining of the stomach orintestine.

Suitable nontoxic pharmaceutically acceptable carriers, and especiallyoral carriers, will be apparent to those skilled in the art ofpharmaceutical and especially oral or peroral pharmaceutical formations.For those not skilled in the art, reference is made to the text entitled"REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporatedherein by reference. Obviously, the choice of suitable carriers willdepend on the exact nature of the particular dosage form, e.g., liquiddosage form [e.g., whether the composition is to be formulated into asolution, a suspension, gel or another liquid form, or solid dosage form[e.g., whether the composition is to be formulated into a pill, tablet,capsule, caplet, time release form or liquid-filled form].

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the antigen (PspA).Minor amounts of other ingredients such as pH adjusters (e.g., a basesuch as NaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents, jelling agents, (e.g., methylcellulose),colors and/or flavors may also be present. The compositions can beisotonic, i.e., it can have the same osmotic pressure as blood andlacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions must be selected to be chemically inert with respect toPspA. This will present no problem to those skilled in chemical andpharmaceutical principles, or problems can be readily avoided byreference to standard texts or by simple experiments (not involvingundue experimentation), from this disclosure.

The immunologically effective compositions of this invention areprepared by mixing the ingredients following generally acceptedprocedures. For example the selected components may be simply mixed in ablender, or other standard device to produce a concentrated mixturewhich may then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity. Generally the pHmay be from about 3 to 7.5. Compositions can be administered in dosagesand by techniques well known to those skilled in the medical andveterinary arts taking into consideration such factors as the age, sex,weight, and condition of the particular patient or animal, and thecomposition form used for administration (e.g., solid vs. liquid).Dosages for humans or other mammals can be determined without undueexperimentation by the skilled artisan, from the Examples below (e.g.,from the Examples involving mice).

When CT is used as an adjuvant for oral immunizations, specific IgAantibodies are induced in secretions. Strong circulatory immuneresponses can also be induced, with IgG and IgA antibodies in the serum,and IgG and IgA antibody-secreting cells in the spleen. The circulatory(or systemic) immune responses elicited by oral (peroral; intragastric)administration of PspA along with CT are comparable with, or evenstronger than, those induced by the administration of similar immunogensby the intragastric (i.g., peroral) route (Wu et al., 1993; Russell etal. 1991). Accordingly, it appears that oral (peroral, i.g.)immunization is an effective route for stimulating common mucosalresponses as well as circulatory antibody responses and can require lessantigen than other immunization routes.

Most soluble or non-replicating antigens are poor immunogens, especiallyby the peroral route, probably because they are degraded by digestiveenzymes and have little or no tropism for the GALT. A notable exceptionis CT, which is a potent mucosal immunogen (Elson et al., 1984),probably because of the G_(M1) ganglioside-binding property of thisbinding subunit, CTB, that enables it to be taken up by the M cells ofPeyer's patches and passed to the underlying immunocompetent cells. Inaddition to being a good mucosal immunogen, CT is a powerful adjuvant(Elson et al., 1989; Lycke et al., 1986; Wilson et al., 1989). Whenadministered in μg does, CT greatly enhances immunogenicity of othersoluble antigens co-administered with it.

CTB is a strong adjuvant when given orally or perorally (e.g.,intragastrically) in mice along with antigen, but CTB has no directadjuvant effect, but can act synergistically with CT (Wilson et al.,1990).

As discussed above, the mechanisms by which CT and CTB act as adjuvantsare not fully understood, but are certainly complex, and appear todepend on several factors, including: 1) the toxic activity associatedwith the ADP-ribosylating property of the A1 subunit (Lycke et al.,1992; Abbas et al., 1991); 2) increased permeability of mucosae (refs.46, 47, 48, 49). This last point is controversial, however, as the invitro effects of CT or CTB on T and B cells are generally inhibitoryrather than stimulatory (Woogen et al., 1987; Garrone et al., 1993;Haack et al., 1993; Quiding et al., 1991; Czerkinsky et al., 1989;Svernerholm et al., 1984). Nevertheless, numerous reports attest to thein vivo mucosal immunoenhancing effects of CT and of CTB couples toantigens (Lycke et al., 1986; Wilson et al., 1989; Abraham et al., 1991;Szu et al., 1989; Chen et al., 1990; Liang et al., 1988; Hakansson etal., 1994; Anderson et al., 1981; Dagen et al., 1995; Dillard et al.,1994). And, CT can selectively inhibit CD8⁺ cells, and therefore tend toabrogate suppressive effects (Elson et al., 1995).

Although carriage of pneumococci can be maintained for long periods inthe very young and the elderly, it is generally not a permanentcondition. Carriage is much less common in older children and youngadults (Gray et al., 1980; Gray et al., 1981; Hendley et al., 1975;Brimblecombe et al., 1958; Masters et al., 1958). One explanation forthese findings is that carriage may be interfered with by immunity(possibly mucosal immunity) to pneumococci (Gray et al., 1980; Gwaltneyet al., 1975). Most human saliva have IgA antibodies to type 23 capsularpolysaccharide and phosphocholine (an immunodominant determinant ofpneumococcal cell wall teichoic acids) (Russell et al., 1990). It seemslikely, therefore, that human sera would also contain antibodies toother pneumococcal antigens. In the cases of group A streptococci, Mproteins have been shown to be required for colonization in rats, andantibodies to M proteins can protect against colonization of the throat(Bessen et al., 1988; Hollingshead et al., 1993). In mice, the inventorshave shown herein that immunity to PspA can prevent carriage of S.pneumoniae.

Antibodies may be effective against carriage in two ways, namely: 1)they might act at the mucosal surface by opsonizing pneumococci,preventing attachment or surface invasion; 2) they might act viaopsonophagocytosis and killing. This latter mechanism could beespecially important if carriage is dependent on minimal invasion of thenasal mucosal surface. The complement fixing antibodies could preventthe invasion and facilitate the killing of any pneumococci that invadedlocally. Alternatively, complement fixing antibodies might be able toact and the mucosal surface if inflammation causes a sufficient releaseof complement, phagocytes, and possibly serum antibody.

One of these mechanisms might play a role in the observation thatcarriage of H. influenzae can be prevented by an intramuscular vaccine(Barbour et al., 1993). It has recently been reported that significantlevels of H. influenzae polysaccharide-specific IgG and IgA are detectedin secretions of children following immunization with the group bpolysaccharide conjugate vaccine (Chiu et al., 1994; Kauppi et al.,1993).

Existing mouse protection data (Briles et al., 1989; Briles et al.,1981; Lock et al., 1988, Lock et al., 1992) suggests that antibodiesthat can opsonize pneumococci (e.g. those to the capsule) are generallymore protective against sepsis than those that block the activities oftoxins (e.g. pneumolysin) or enzymes (e.g. autolysin or neuraminidase).However, at the mucosal surface, the role played by antibodies thatinactivate toxins and enzymes may be greater than that played by opsonicantibodies. The reason to suspect this is that for opsonic antibodies toexert their anti-bacterial effect, complement and phagocytes arerequired. Phagocytes are rare on the surface of normal nasopharyngealtissue, and even if present, the phagocytes do not have the filteringaction of the spleen and reticuloendothelial system to increase theirchance of interactions with opsonized bacteria. Antibodies that blockthe virulence enhancing effects of pneumolysin and pneumococcal enzymesshould be able to bind their antigens just as effectively whetherphagocytes were present or not.

The results provided herein show that oral or peroral (e.g., i.g.)immunization with heat-killed pneumococci, and pneumococcal lysates, andpurified PspA can protect mice against sepsis and therefore carriagetoo. As noted earlier, the ability of a vaccine to protect againstdisease not only in the immunized host, but, by eliminating carriageamong immunized individuals, the pathogen and hence any disease itcauses may be eliminated from the population as a whole.

The vaccine or immunogenic composition which is administered orally,perorally or intragastrically as provided herein may be formulated inany convenient manner and in a dosage formulation consistent with themode of administration and the elicitation of a protective response (seediscussion above and Examples below). The quantity of antigen to beadministered depends on the subject to be immunized and the form of theantigen. Precise amounts and form of the antigen to be administereddepend on the judgment of the practitioner. However, suitable dosageranges are readily determinable by those skilled in the art from thisdisclosure, without undue experimentation, and may be of the order ofmicrograms to milligrams. Suitable regimes for initial administrationand booster doses also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, withoutundue experimentation.

Further, the invention also comprehends a kit wherein PspA or a fragmentthereof is provided. The kit can include in a container separate fromthe PspA or fragment thereof, a suitable carrier, diluent or excipient.The kit can also include, in a container separate from the PspA orfragment thereof or from the carrier, diluent or excipient, or alreadyin admixture with either of these components, an adjuvant, such ascholera toxin. Thus, the kit comprises: (i) PspA or fragment thereof,(ii) carrier, excipient or diluent, and optionally, (iii) adjuvant,wherein there are separate containers for (i) and (ii), or for (i), (ii)and (iii), or for (i) and the combination of (ii) and (iii), or for (ii)and the combination of (i) and (iii). Additionally, the kit can includeinstructions for mixing or combining ingredients and/or administration.

The following Examples are provided for illustration and are not to beconsidered a limitation of the invention.

EXAMPLES Example 1

Characterization of Immune Responses in Mice Orally Immunized with PspA

Prior to oral immunization, mice were deprived of food for two hours andthen given a solution of sodium bicarbonate to neutralize stomach acid.Thirty minutes later, mice were orally-immunized by gastric intubationwith 7.5 μg of purified native PspA with or without CT. Oralimmunizations were carried out on day 0, 7, 14, and 35. Serum and fecalextracts were collected prior to immunization on days 7, 14 and 35. Seraand fecal pellets were also collected on days 21, 28, 42 and 49. Seraand fecal extracts were obtained and assayed as previously described(Jackson R. J., K. Fujihoski, J. Xu-Amano, H. Kiyono, C. O. Elson and J.R. McGhee. 1993. "Optimizing oral vaccines: induction of systemic andmucosal B. cell and antibody responses to tetanus toxoid by use ofcholera toxin as an adjuvant", Infect. Immun. 61:4272-4279 de Vos etal., 1991). Additionally, the ability of oral immunization with PspA toelicit protective immunity was examined using the previously describedmouse model of pneumococcal sepsis (Briles et al., 1992; Briles et al.,1989).

When mice were orally immunized with PspA alone, antigen-specific serumIgG responses were detected after the primary immunization. The Log2titers were 5 to 7 (FIG. 1). These responses were not enhanced followingthe booster oral immunizations, and no IgM responses were detectedduring the analysis period (FIG. 1). No IgA antibody to PspA wasdetected until day 49 when the titer was only Log2-5. The fecal extractshad no detectable levels of antigen-specific responses.

When PspA was given orally with CT, antigen specific IgM, IgG, and IgAresponses were induced in the serum. PspA-specific IgM antibodies Log2=9were detected after the second injection. PspA-specific IgG responseswere elevated to Log2=19 after the 4th oral immunization with PspA plusCT; Also, PspA-specific serum IgA responses of Log2=9 and 13 weredetected on day 42 and 49, respectively (FIG. 2). Mucosal IgA responsesin fecal extracts (Log2=5) were detected after the fourth immunization.

Example 2

Characterization of Protection Against Sepsis Elicited by Mice ImmunizedOrally with PspA

Mice were challenged intravenously with 3.6×10³ colony forming units(CFU) of S. pneumoniae A66 two weeks following the 4th oral immunizationwith PspA alone or PspA plus CT. The results (Table 1, below)demonstrated that oral immunization with purified PspA (using CT as anadjuvant) can provide systemic protective immunity to otherwise fatalinfections with S. pneumoniae.

Oral immunization with PspA using cholera toxin as an adjuvant inducesprotective immunity to systemic challenge with capsular type 3 strainA66 Streptococcus pneumoniae.

    ______________________________________                                        Antigen used for immunization                                                               PspA + CT   PspA      None                                      ______________________________________                                        Log.sub.2 Reciprocal titer of                                                               19          6         <4                                          IgG anti-PspA                                                                 Day of Death >21,>21,>21,>21 1,1,2,3,4, 1,1,2,2,3                             for individual mice >21,>21,>21 >21,>21,>21                                   Median Day of Death >21 3.5 2                                                 Alive:Dead 7:0 3:5 0:5                                                        P value vs. none 0.003 n.s.                                                   P value vs. "PspA" 0.05  n.s.                                               ______________________________________                                         Note: C57BL/6J Mice were challenged intravenously with 3.5 ×            10.sup.3 A66. P values shown above were calculated by the two tailed          Wilcoxon twosample rank test. Using the KruskalWallis nonparametric           analysis of variance the P value is 0.0071.                              

Example 3

Induction of Protective Humoral and System Immunity in Mice Immunizedwith PspA

For systemic immunization of mice 0.5 μg of purified, native PspA withComplete Freund Adjuvant (CFA) was injected subcutaneously on days 0 and14 (at a ratio of 1:1 CFA to PspA in PBS). For oral immunication, micewere deprived of food for two hours, and then administered by gastricintubation a solution of sodium bicarbonate to neutralize stomach acidprior to the immunization. Thirty minutes later, mice wereorally-immunized by gastric intubation with 7.5 μg of purified, nativePspA, with or without mucosal adjuvant cholera toxin (CT). Oralimmunizations were carried out on day 0, 7, 14 and 35. A total of sixmice were used in each control and experimental group.

Serum and fecal extracts were prepared as previously descrived (Jackson,R. J. et al, Infect. Immun. 1993) 61: 4272-79). Serum and fecal extractswere collected weekly intervals and assayed for PspA-specific IgM, IgGand IgA antibodies by ELISA. 96 well plates (Nunc Immuno Plate,Roskilde, Denmark) were coated with PspA in PBS for overnight at 4° C.After washing 3 times with PBS, wells were blocked for 1 hours with PBScontaining 10% goat serum. Ser. two-fold dilutions of samples weretested, starting at a dilution of 1:2 (fecal extract) and 1:32 (serum).Plates were incubated for 4 hours at room temperature. After incubation,captured antibodies were detected with biotinylated anti-mouse IgM, IgGand IgA (1 mg/ml, Southern Biotechnology Associates (SBA), Birmingham,Ala.) and horseradish peroxidase (HRP)-streptoavidin (0.5 mg/ml, Gibco,Gaithersburg, Md.). ABTS (2,2 azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), diamonium salt; Sigma, St.Louis, Mo.) was used for color development. Titres were recorded as thelast dilution which gave an absorbance (414 nm) of 0.1 units greaterthan control serum from non immunized mice.

To examine the antigen-specific fecal IgA responses, the method ofluminometry was applied. Briefly, 96 well plates (Dynatech Microlite®,Chantilly, Va.) were prepared exactly as described for ELISA. Followingthe incubation of the secondary biotinylated antibody, plates werewashed with PBS-Tween containing 2 mM EGTA, added streptaequorin andincubated 1 hour at room temperature. The plates were washed, dried andplaced in the luminometer for the color development. The wellscontaining the aequorin derivatives were developed in a Dynatech ML-3000luminometer by injection of 50 mM Tris-HCl, pH 7.5, containing 2 mMcalcium acetate. Results were expressed as relative light unit (RLU)following subtraction of blank values. To determine endopoint titer forthe luminometric assay, the straight line generated following backgroundsubstraction was extrapolated (E titer) to a baseline 1000 relativelight units above background.

To examine the number of PspA-specific antibody forming cells (AFCs),ELISPOT assay was performed as previously described (Jackson, R. J. etal., Infect. Immun. (1993) 61: 4272-79). Single cell suspensions wereprepared from spleen by mechanical dissociation. Ninety-six wellnitrocellulose plates (Millititer HA; Millipore Corp., Bedford, Mass.)were used for the assay. The plates were coated with 0.5 mg of purified,native PspA. After incubation overnight at 4° C. plates were washed andblocked with 10% FCS-RPMI 1640. The blocking solution was discarded, and100 ml of cells in 10% FCS-RPMI 1640 at various dilutions were added.Cell suspensions were incubated for 4 hours at 37° C. in 5% CO₂atmosphere. Plates were washed with PBS and PBS-0.05% Tween-20. Thesecondary antibody solution consisted of 100 ml of a 1:1,000 dilution ofgoat anti-mouse IgM, IgG and IgA conjugated to horseradish peroxidase(Southern Biotechnology Associates, Birmingham, Ala.) in PBS-0.05%Tween-20 containing 1% BSA. Following overnight incubation, plates werewashed with PBS, and developed by the addition of 200 ml of3-amino-9-ethylcarbazole dissolved in 0.1M sodium acetate buffercontaining H₂ O₂ per well. Plates were incubated at room temperature for15-20 min and washed with water, and AFCs were counted with the aid of astereomicroscope.

Mice which were subcutaneously immunized with 0.5 μg of purified, nativePspA with CFA were used in order to establish a base line for systemicimmunization. PspA-specific IgG responses were detected by 14 days afterthe primary immunization and levels of antigen-specific titers wereincreased following secondary immunization. There PspA-specific IgGresponses were maintained for greater than 70 days (FIG. 3). In order toconfirm the source of these PspA-specific IgG antibodies, mice weresacrificed 4 days after secondary immunization. Splenic mononuclearcells were isolated and assayed for the detection of PspA-specificantibody producing cells by the ELISPOT assay. A significant number ofantigen-specific IgG producing cells were detected in splenocytesisolated from mice subcutaneously immunized with PspA when compared withcontrol group (unimmunnized mice). However, IgM and IgA producing cellswere very low or undetectable (FIG. 4). These results support theconclusion that systemic immunization of PspA induces high levels ofantigen-specific serum responses.

When mice were orally-immunized with purified, native PspA alone,antigen-specific serum IgG responses were elicited after primaryimmunization, however, the titer of responses was low (1:32-1:128) whencompared with the systemic immunization (FIGS. 3 and 5A), and 3 out of 8mice were protected from a lethal challenge with S. pneumoniae. Further,those responses were not enhanced following the booster oralimmunization. Soluble proteins are generally not strong immunogens whengiven by the oral route. This result, however, suggests that smallamounts of PspA are sufficient to induce serum responses by the oralroute which are able to protect against challenge. Very small IgAresponse (1:32) were detected 3 weeks after 4th oral immunization on day35. No antigen-specific IgM responses were detected during the analysisperiod following immunization (FIG. 5A). In the case of fecal extracts,there was no detectable levels of antigen-specific immune responses.

Oral administration of mucosal adjuvant CT with soluble protein antigennot only induces antigen-specific serum responses, but it also is aneffective immunization regimen for the generation of antigen-specificantibody responses in mucosal sites. Thus, when purified, native PspAwas given orally with 10 μg CT on day 0, 7, 14 and 35, antigen-specificIgM, IgG and IgA responses were induced in the serum. For example, aftersecondary immunization, PspA-specific IgM antibodies were induced(1:512) and shifted to secondary type responses where high levels of IgGresponses (1:16,384) were elicited on day 21 (FIG. 5B). ThesePspA-specific IgG responses were elevated up to 1:524,288 after 4th oralimmunization with PspA plus CT. Furthermore, PspA-specific IgA serumresponses were detected on day 42 and 49 (FIG. 5B). When those mice werechallenged with a lethal dose of S. pneumoniae intravenously, all micesurvived challenge (Table 2).

In addition, antigen-specific IgA antibodies were detected in fecalextracts obtained from 2 weeks after the third oral immunization and theresponses were increased following the 4th immunization (FIG. 6). Whensubclass of PspA-specific IgG response was evaluated, main response wasnoted as IgG1 followed by IgG2b (FIG. 7). Further, IgA anti-PspAappeared in the fecal extracts following the third immunization, and theresponse was increased after the fourth immunization. In this regard, CTis known to selectively induce antigen-specific Th2 type response whichpromotes IgG1 in serum as well as mucosal IgA responses when it wasgiven orally together with protein antigen.

Thus, these results showed that oral immunization with PspA plusadjuvant CT lead to the generation of antigen-specific Th2 typeresponses accounting for the induction of PspA-specific IgG1 as well asS-IgA responses in systemic and mucosal sites, respectively. Takentogether, these results showed that oral immunization of PspA with CT asmucosal adjuvant was capable of inducing PspA-specific serum as well asmucosal antibody responses.

Example 4

Oral Immunization with PspA and Cholera Toxin in Order to EstablishProtective Humoral Immunity Against Pneumonoccal Infection

The pneumococcal in vivo protection analysis was performed as previouslydescrived (McDaniel, L. S., et al. Infect. Immun. (1991) 59: 222-228).Briefly, fourteen days after fourth immunization with PspA alone orconjugated to cholera toxin (CT), mice were challenged intravenouslywith 3.6×10³ CFU of pneumococcal strain A66. The survival of mice wasmonitered for 21 days. The mice orally-immunized with PspA plus CTshowed antigen-specific serum IgG titer of 19 prior to the challenge(Table 2, below). In the mice orally-immunized with PspA alone, thelevels of antigen-specific IgG response was low (serum titer of 6). Whenthese mice were systemically challenged with a lethal dose of A66strain, oral immunization with PspA and CT protected all the mice fromthe infection. However, oral immunization with PspA alone failed toprovide complete protection, and three out of eight mice survivedchallenge (Table 2). These results demonstrated that oral immunizationwith PspA can provide protective immunity and mucosal adjuvant CT canfurther elicit protective humoral immune responses against pnemococcalinfection.

Hence, oral immunization with PspA vaccine can induce a protective,humoral immune response against S. pneumoniae infection. Further, sinceit has been shown that PspA elicits cross protection against differentcapsular serotypes, oral immuization with PspA plus CT could induce aprotective level of systemic responses against different capsularserotypes of pneumococcal infection.

                  TABLE 2                                                         ______________________________________                                        Oral immunization with PspA plus CT induces protective                          immunity to systemic challenge with capsular type 3                           strain A66 Streptococcus pneumoniae                                                     Serum IgG                                                            Titer Day of Death Median Day P value                                        Vaccine (1/log2) After Challenge of Death vs. None*                         ______________________________________                                        PspA + CT                                                                             19        >21,>21,>21,>21                                                                           >21     0.003                                       >21,>21,>21                                                                 PspA 6 1,1,2,3,4,>21 3.5 N.S                                                    >21,>21                                                                     None <4 1,1,2,2,3 2 N.A                                                     ______________________________________                                         Note: The mice were challenged intravenously with 3.6 × 10.sup.3 CF     of A66.                                                                       *P values shown above were calculated by the two tailed Wilcoxon twosampl     tank test using, N.S: not significant, N.A: not applicable.              

These results are analogous to providing humans with solid orsolid-with-liquid-center forms which are absorbed primarily in the gut(i.e., swallowed whole for intragastric absorption), and are alsoindicative of the utility of liquid forms (which may be moreadvantageous for administration to children, especially children age 2or less).

In summary of this disclosure, the present invention provides a methodof immunizing a host against pneumococci in a host and for protectingagainst systemic and humoral infection by pneumococci, by oral,particularly intragastric, administration of PspA in various forms.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forthabove, as many apparent variations thereof are possible withoutdeparting from the spirit or scope thereof.

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What is claimed is:
 1. A method of immunization of a host againstpneumococcal infection, which comprises orally administering to the hostan immunizing amount of a composition comprising a pneumococcalcomponent wherein said pneumococcal component consists essentially ofisolated pneumococcal surface protein A (PspA) or at least one isolatedfragment of such PspA containing at least one protection-elicitingepitope.
 2. The method of claim 1 wherein said PspA is an isolated andpurified PspA.
 3. The method of claim 1 wherein said compositionincludes an adjuvant.
 4. The method of claim 3 wherein the adjuvant is acholera toxin.
 5. The method of claim 1 wherein said oral administrationis effected intragastrically.
 6. A method of immunization of a hostagainst Streptococcus pneumoniae, which comprises intragastricallyadministering to the host an immunizing amount of a compositioncomprising a pneumococcal component wherein said pneumococcal componentconsists essentially of isolated and purified PspA, or an isolated andpurified immunogenic fragment thereof.
 7. The method of claim 6 whereinsaid composition includes an adjuvanting amount of a cholera toxin. 8.The method of claim 6 wherein the composition includes an adjuvant.
 9. Amethod for eliciting an immunological response in a host against S.pneumoniae which comprises orally administering to the host an effectiveamount of a composition comprising a pneumococcal component wherein saidpneumococcal component consists essentially of isolated pneumococcalsurface protein A (PspA) or at least one isolated fragment of such PspAcontaining at least one immunologically active epitope.
 10. The methodof claim 9 wherein the orally administering is performed byintragastrically administering.